SUMMARY CRISPR-Cas systems are diverse adaptive immunity modules, widespread across bacteria and archaea. We study Type I CRISPR-Cas systems, which represent the majority of all systems. We use the Escherichia coli Type I-E system as a model. Importantly, many aspects of Type I CRISPR systems are mechanistically distinct from the well-studied Cas9 Type II system. Our long-term goal is to develop a detailed mechanistic understanding of all steps of E. coli CRISPR-Cas function, including CRISPR RNA expression, targeting of invading DNA molecules, and acquisition of new immunity elements (?spacers?). A complete understanding of CRISPR systems is a highly significant goal, since they have huge potential to be developed as research tools for genome engineering and high-throughput functional genomic analyses, and as sequence-specific anti-microbials. The two major aspects of Type I CRISPR-Cas biology are (i) targeting of invading DNA molecules and (ii) acquisition of new spacers. The Cascade protein complex binds CRISPR RNAs and targets DNA sequences that are complementary to the CRISPR RNA sequence. This results in destruction of the target DNA by the Cas3 nuclease. The major mechanism by which new spacers are acquired is known as ?priming?. Priming requires that Cascade (bound to a CRISPR RNA) targets an invading DNA molecule, although not necessarily with high affinity. This results in acquisition of a spacer from a different position in the targeted DNA molecule. Key questions about Type I CRISPR-Cas biology remain unanswered. First, it is unclear how the processes of Cascade binding to target DNA, Cas3-mediated destruction of target DNA, and priming, are connected. Second, it is unclear which sequence elements are required for each of these processes. Third, the process of priming is poorly understood. We do not know the composition of the priming complex, nor do we know how new spacers are acquired. We will leverage our expertise in genetics, genomics, and high-throughput techniques to address these critical, unanswered questions by pursuing the following specific aims: Specific Aim 1 ? Determine the spacer/protospacer sequence requirements for Cascade binding, priming and target destruction. Specific Aim 2 ? Determine the molecular details of priming. Specific Aim 3 ? Determine the role of Nus factors in CRISPR RNA expression.